The Peptide KLVFF-K6 Promotes β-Amyloid(1-40) Protofibril Growth by Association but Does Not Alter Protofibril Effects on Cellular Reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT)

Melissa A. Moss, Michael R. Nichols, Dana Kim Reed, Jan H. Hoh, Terrone L. Rosenberry

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

The peptide KLVFF-K6 was observed by Lowe et al. (Biochemistry 40:7882-7889, 2001) to simultaneously enhance amyloid β-protein (Aβ) fibrillogenesis and decrease cellular toxicity, as measured in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. It was postulated that accelerated Aβ aggregation and precipitation induced by KLVFF-K6 may lead to an increase in less toxic insoluble fibrils at the expense of more toxic soluble protofibrils. In a previous study, we distinguished between two modes of protofibril growth: elongation by monomer deposition and direct protofibril-protofibril association. These growth mechanisms could be resolved by varying Aβ monomer and NaCl concentrations. Using assays designed to isolate these distinct modes of protofibril growth, we report here that larger Aβ aggregates formed in the presence of KLVFF-K6 resulted from enhanced protofibril association. 3H-Radiomethylated KLVFF-K6 bound to associated protofibrils with an apparent Kd of 180 nM, and concentrations of free [3H]KLVFF-K6 in this range were sufficient to convert soluble protofibrils to sedimentable fibrils. However, promotion of Aβ protofibril association by KLVFF-K6 had no effect on Aβ-induced decreases in cellular MTT reduction. Therefore, our data do not support the proposal that insoluble fibrils formed with KLVFF-K6 are less toxic than soluble protofibrils. KLVFF-K6 did not alter rates of protofibril elongation by monomer deposition. In contrast, when added to Aβ monomers isolated with the use of size-exclusion chromatography, KLVFF-K6 inhibited fibrillogenesis, as measured by thioflavin T fluorescence, and this inhibition was paralleled by a failure to alter cellular MTT reduction.

Original languageEnglish (US)
Pages (from-to)1160-1168
Number of pages9
JournalMolecular Pharmacology
Volume64
Issue number5
DOIs
StatePublished - Nov 2003

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Amyloid
Peptides
Growth
Poisons
amyloid beta-protein (16-20)
thiazolyl blue
Serum Amyloid A Protein
Biochemistry
Gel Chromatography
Fluorescence

ASJC Scopus subject areas

  • Pharmacology

Cite this

The Peptide KLVFF-K6 Promotes β-Amyloid(1-40) Protofibril Growth by Association but Does Not Alter Protofibril Effects on Cellular Reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT). / Moss, Melissa A.; Nichols, Michael R.; Reed, Dana Kim; Hoh, Jan H.; Rosenberry, Terrone L.

In: Molecular Pharmacology, Vol. 64, No. 5, 11.2003, p. 1160-1168.

Research output: Contribution to journalArticle

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abstract = "The peptide KLVFF-K6 was observed by Lowe et al. (Biochemistry 40:7882-7889, 2001) to simultaneously enhance amyloid β-protein (Aβ) fibrillogenesis and decrease cellular toxicity, as measured in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. It was postulated that accelerated Aβ aggregation and precipitation induced by KLVFF-K6 may lead to an increase in less toxic insoluble fibrils at the expense of more toxic soluble protofibrils. In a previous study, we distinguished between two modes of protofibril growth: elongation by monomer deposition and direct protofibril-protofibril association. These growth mechanisms could be resolved by varying Aβ monomer and NaCl concentrations. Using assays designed to isolate these distinct modes of protofibril growth, we report here that larger Aβ aggregates formed in the presence of KLVFF-K6 resulted from enhanced protofibril association. 3H-Radiomethylated KLVFF-K6 bound to associated protofibrils with an apparent Kd of 180 nM, and concentrations of free [3H]KLVFF-K6 in this range were sufficient to convert soluble protofibrils to sedimentable fibrils. However, promotion of Aβ protofibril association by KLVFF-K6 had no effect on Aβ-induced decreases in cellular MTT reduction. Therefore, our data do not support the proposal that insoluble fibrils formed with KLVFF-K6 are less toxic than soluble protofibrils. KLVFF-K6 did not alter rates of protofibril elongation by monomer deposition. In contrast, when added to Aβ monomers isolated with the use of size-exclusion chromatography, KLVFF-K6 inhibited fibrillogenesis, as measured by thioflavin T fluorescence, and this inhibition was paralleled by a failure to alter cellular MTT reduction.",
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